The goal of the proposed research is to develop novel metal-free click chemistry pretargeted approaches for cancer radioimmunotherapy (RIT). It is hypothesized that novel in vivo cycloaddition chemistry will greatly facilitate the use of pretargeted antibodies as cancer treatment agents, overcoming their relatively slow in vivo pharmacokinetic properties and yielding high tumor to normal tissue localization. The monoclonal antibody (mAb) CC49 will be used as the targeting agent. CC49 binds the-TAG72 mucin antigen that is over expressed on a number of cancers including colon carcinomas. The CC49 mAb will be derivatized with trans-cyclooctene (TCO) and used to target the tumor prior to injection of the therapeutic probe in a 2-step pretargeting approach. A bis(pyridinyl)tetrazine (tetrazine) conjugated metal will be radiolabeled with the ?-emitter 212Bi or its parent 212Pb or the -emitter 177Lu and injected hours to a few days post injection of the tumor-avid antibody. In vivo, a rapid and highly specific cycloaddition reaction will occur between the TCO on the tumor- targeted antibody and the tetrazine moiety of the radiolabeled chelator-tetrazine yielding a covalent conjugate. Unreacted radiolabeled chelator-tetrazine will be rapidly cleared from the body resulting in a high tumor to normal tissue localization of radioactivity. The aims of the proposed research are as follows. Aim1, pretargeting chemistry development, which encompassed second generation cyclooctene, chelator-tetrazine conjugate and clearing agent synthesis and characterization. Aim 2, radiochemistry and conjugation studies, will focus on the radiochemical labeling efficiencies and radiochemical stabilities of the chelator-tetrazine conjugates that will be radiolabled with 212Bi, 212Pb and 177Lu. The bioacitivties of TCO-CC49 and the labeled tetrazine-chelators will be evaluated with TAG72 expressing cell lines. Aim 3, evaluation of new pretargeting components, will optimize the dose to the tumor while reducing the dose to normal tissues, including blood kinetics and biodistribution of newly developed tetrazine probes. Aim4, therapy studies, will take the best combination of ? and emitter pretargeting molecules and examine their therapeutic efficacy in a colon cancer xenograft mouse model. Milestones for the project include, improving in vivo cycloaddition reaction by 10 fold, targeting a greater that five-fold improvement in dose to tumor versus normal tissue using pretargeted RIT and a five-fold improvement in targeted ? RIT. The final milestone will be to demonstrate that pretargeted therapy studies will yield statistically significantly extension in survival in a mouse colon cancer model. This application of novel in vivo cycloaddition chemistry in a pretargeted approach for cancer radiotherapy is highly innovative. The cycloaddition reaction of olefins with tetrazines in vivo is the first of its kind to be used effectively at clincally relevant conditions in vivo due to its unprecedented reactivity, and for the first time has the potential to extend this type of organic chemistry in to man. This project would be the first of it kind in vivo application of TCO-tetrazine cycloaddition chemistry with pretargeted ?-particle radiotherapy.